Coenzyme specificity of D-amino acid oxidase for the flavin moiety of fad

Abstract

Summary Analogues of flavin—adenine dinucleotide have been synthesized from pyridi-nium salts of various flavin phosphate esters and N,N ′-dicyclohexylcarboxamidinium adenosine 5′-phosphoromorpholidate in pyridine with o -chlorophenol or benzyl alcohol. The behavior of these riboflavin-replaced analogues of flavin-adenine dinucleotide with D -amino acid oxidase ( D -amino acid:O 2 oxidoreductase (deaminating), EC 1.4.3.3) has led to the following conclusions about the coenzyme specificity of the enzyme: 1. A polyol side chain in the flavin is required for maximum activity, since 2′-deoxyriboflavin-adenine dinucleotide and 5′-hydroxypentylflavin-adenine dinucleotide are significantly less active than flavin-adenine dinucleotide. 2. A flavin side chain of five carbons in length is necessary for coenzymatic activity, although apparently less necessary for binding to the apoenzyme, since D -erythroflavin-adenine dinucleotide and 4′-hydroxybutylflavin-adenine dinucleotide are competitive inhibitors of flavin-adenine dinucleotide. 3. Substitutions in the isoalloxazine system which give rise to alterations in the oxidation-reduction potential of the flavin lower coenzymatic activity, since 6,7-dichlororiboflavin-adenine dinucleotide is less active than flavin-adenine dinucleotide. 4. The 3-nitrogen in the isoalloxazine ring cannot be alkylated without total loss of coenzymatic activity and considerable loss of ability to bind, as 3-methylribo-flavin-adenine dinucleotide is a relatively weak inhibitor. Overall, the results indicate considerable specificity for the flavin moiety of flavin-adenine dinucleotide by D -amino acid oxidase, though somewhat less narrow than that seen for the adenylate moiety.